Mariko Sashika

The Phylogenetic Position of Anaplasma bovis and Inferences on the Phylogeny of the Genus Anaplasma

ABSTRACT The present study aimed to determine the complete citrate synthase (gltA) and heat-shock protein (groEL) gene sequences of Anaplasma bovis and to infer phylogenetic relationships within the genus Anaplasma. Multiple alignments from single and concatenated sequences of the 16S rRNA, gltA and groEL genes of the genus Anaplasma were subjected to phylogenetic analyses. Percent identities of A. bovis nucleotide sequences were found highest with A. phagocytophilum in gltA (65.4%) and groEL (79.8%). Single gene phylogenetic tree results assumed similar phylogenetic positions within the genus Anaplasma, except for A. bovis. However, consensus and concatenated sequence phylogenetic trees showed similar results, revealing 2 subgroups within the genus.

A relapsing fever group Borrelia sp. similar to Borrelia lonestari found among wild sika deer (Cervus nippon yesoensis) and Haemaphysalis spp. ticks in Hokkaido, Japan

A relapsing fever Borrelia sp. similar to Borrelia lonestari (herein referred to as B. lonestari-like) was detected from wild sika deer (Cervus nippon yesoensis) and Haemaphysalis ticks in the eastern part of Hokkaido, Japan. The total prevalence of this Borrelia sp. in tested deer blood samples was 10.6% using conventional PCR and real-time PCR. The prevalence was significantly higher in deer fawns compared to adults (21.9% and 9.4%, respectively). Additionally, there was significant regional difference between our two sampling areas, Shiretoko and Shibetsu with 17% and 2.8% prevalence, respectively. Regional differences were also found in tick species collected from field and on deer. In the Shiretoko region, Haemaphysalis spp. were more abundant than Ixodes spp., while in Shibetsu, Ixodes spp. were more abundant. Using real-time PCR analysis, B. lonestari-like was detected from 2 out of 290 adult Haemaphysalis spp. ticks and 4 out of 76 pools of nymphs. This is the first report of a B. lonestari-like organism in Haemaphysalis spp. ticks, and the first phylogenetic analysis of this B. lonestari-like organism in Asia. Based on our results, Haemaphysalis spp. are the most likely candidates to act as a vector for B. lonestari-like; furthermore, regional variation of B. lonestari-like prevalence in sika deer may be dependent on the population distribution of these ticks.

Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan

A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan.

Dynamics, co-infections and characteristics of zoonotic tick-borne pathogens in Hokkaido small mammals, Japan

Many of the emerging infectious diseases originate in wildlife and many of them are caused by vector-borne pathogens. In Japan, zoonotic tick-borne pathogens (TBPs) are frequently detected in both ticks and wildlife. Here, we studied the infection rates of potentially zoonotic species, including Anaplasma, Ehrlichia, Neoehrlichia and Babesia spp., in Hokkaidos most abundant small mammals as they relate to variable extrinsic factors that might affect the infection rates of these pathogens. A total of 412 small mammals including 64 Apodemus argenteus, 219 Apodemus speciosus, 78 Myodes rufocanus, 41 Myodes rutilus, 6 Myodes rex and 4 Sorex unguiculatus were collected from Furano and Shari sites in Hokkaido, Japan, in 2010 and 2011 and were examined by multiplex PCR for TBPs. A reverse line blot hybridization (RLB) was then developed for the specific detection of 13 potentially zoonotic TBPs. A total of 4 TBPs were detected: Anaplasma sp. AP-sd, Ehrlichia muris, Candidatus Neoehrlichia mikurensis and Babesia microti. The infection rates were 4.4% (18/412), 1.2% (5/412), 13.1% (54/412) and 17.2% (71/412), respectively. The infection rates of each of the detected TBPs were significantly correlated with host small mammal species. A total of 22 (two triple and 20 double) co-infection cases were detected (5.3%). The most frequent co-infection cases occurred between Candidatus N. mikurensis and B. microti 68.2% (15/22). Further studies are required to examine human exposure to these zoonotic TBPs in Hokkaido.

Molecular identification and characterization of piroplasm species in Hokkaido sika deer (Cervus nippon yesoensis), Japan

Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido.

First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan

Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.

Development and evaluation of an interferon-γ release assay in Asian elephants (Elephas maximus)

We developed an interferon-γ release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-γ (rEIFN-γ) as well as native interferon-γ from the Asian elephants was performed using anti-elephant IFN-γ rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-γ rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-γ in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.

Timing of Puberty and Its Relationship with Body Growth and Season in Male Raccoons (Procyon lotor) in Hokkaido

Abstract The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.

Genetic Diversity and Population Structure of East Asian Raccoon Dog (Nyctereutes procyonoides): Genetic Features in Central and Marginal Populations

The raccoon dog (Nyctereutes procyonoides) is endemic to East Asia but has been introduced in Europe. Its high adaptability enabled its rapid colonization of European countries, where population growth has been raising concerns regarding ecosystem disturbance and the spread of zoonotic diseases. The genetic diversity and structure of endemic, source, and introduced populations from seven locations across South Korea, China, Russian Far East, Finland (spread to Finland after introduction to European part of Russia from Russian Far East), Vietnam, and Japan (Honshu and Hokkaido) were examined based on 16 microsatellite loci. Two major and significantly different (FST = 0.236) genetic clusters were found: continental (South Korean, Chinese, Russian, Finnish, and Vietnamese) and island (Japanese) populations. The continental raccoon dog population comprises three subpopulations (Chinese_Russian_Finnish, South Korean, and Vietnamese) and the Japanese population consists of Honshu and Hokkaido subpopulations. The genetic diversity and geographic structure of raccoon dogs in East Asia has been influenced by natural barriers to gene flow and reveals a typical central-marginal trend in genetic diversity (continental vs. island, and central vs. marginal or source vs. introduced within continental populations). The detected differences between continental and island populations agree with those reported in previous studies that considered these populations as different species.

Sex-biased natal dispersal in Hokkaido brown bears revealed through mitochondrial DNA analysis

Understanding natal dispersal patterns is fundamental in the ecology and conservation biology of large wild carnivores. In this study, we used two approaches to determine genetic variation and dispersal patterns of brown bears in the Shiretoko Peninsula, eastern Hokkaido, Japan. The first approach was a large-scale genetic analysis. We analyzed haplotypes from the mitochondrial DNA (mtDNA) control region of 760 individual samples collected throughout the peninsula during 1998–2016. We detected seven haplotypes, including two that were confirmed for the first time. In females, the distribution of haplotypes was geographically structured, whereas haplotypes in males were distributed widely throughout the peninsula. Only some males in the lower peninsula had haplotypes that were not detected within the peninsula. The second approach was a local-scale genetic analysis, including intensive focal sampling in the Rusha area, a special wildlife protection area on the peninsula. Proportions of mtDNA haplotypes in adult bears were investigated and compared between the sexes. Although more than half of the females had the same haplotype, males had more diverse haplotypes, suggesting that they came to the Rusha area from other regions. Thus, our study revealed that mtDNA haplotype distribution has been maintained by female philopatry, and that bears exhibit male-biased dispersal. Furthermore, the lower peninsula appears to act as a contact zone between the peninsula and mainland Hokkaido, which is important for maintaining genetic diversity.